Protein structure, amino acid composition and sequence determine proteome vulnerability to oxidation-induced damage

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation‐resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ‐irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein‐intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties

A Comparative Study on Hepatitis C Predictions Using Machine Learning Algorithms

Hepatitis C virus (HCV) is known to be the major cause of chronic liver disease. Based on research, HCV has caused more than 100.000 cases of liver cancer per year. This virus has become the cause of at least 280.000 deaths. To diagnose HCV, it takes at least two different tests, namely serological assays and molecular tests, which are quite costly and complex. With Machine Learning technology, the diagnosis of any disease or virus can be made by detecting different patterns or relationships. Therefore, this study aims to predict the Hepatitis C virus using different machine learning algorithms and find out the best model for the classification of Hepatitis C disease. Furthermore, this study shows some visualizations to find out the relationships between attributes. We used different machine learning algorithms, namely K-Nearest Neighbour, Support Vector Machine, Random Forest, Neural Network, Naïve Bayes, and Logistic Regression. The performance of those different machine learning algorithms was evaluated using four different metrics, which are classification accuracy, precision, recall, and F-1 score. The classification accuracy results are 96.5%, 96.7%, 97.3%, 97.1%, 96%, 97.9% each for k-NN, SVM, RandomFores, Neural Network, Naïve Bayes and Logistic Regression. Based on the results, each model showed high performance, but Logistic Regression performs the best result. With the results conducted by this study, it is hoped that it can help the diagnosis process of HCV based on laboratory data. However, it is important to communicate the shortcomings and some possible improvements for each model. Keywords: Machine Learning, Predictions, Hepatitis C Viru

Italian Proteomics Association, 5th Annual National Conference Abstract Volume

Abstract volume of the Italian Proteomics Association 5th Annual national conferenc

HIGH RESOLUTION MASS SPECTROMETRIC STRATEGIES IN DRUG DISCOVERY FOR THE INVESTIGATION OF COVALENT AND NON-COVALENT INTERACTIONS

High resolution mass spectrometric strategies in drug discovery for the investigation of covalent and non covalent interactions Dott. Danilo De Maddis PhD tutor: Prof. Giancarlo Aldini The research work here described was focused on the set-up and application of analytical methods for studying compounds effective as inhibitors of the advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) as well as as antagonists of the receptors of AGEs (RAGE). AGEs and ALEs represent a quite complex and heterogeneous class of compounds that are formed by different mechanisms, by heterogeneous precursors and can be formed either exogenously or endogenously. AGEs represent a class of covalently modified proteins generated by oxidative and non-oxidative pathways, involving sugars or their degradation products. The term ALEs includes a variety of covalent adducts which are generated by the non-enzymatic reaction of reactive carbonyl species (RCS), produced by lipid peroxidation and lipid metabolism, with the nucleophilic residues of macromolecules, especially proteins. AGEs and ALEs share some common properties, for example, both consist of non-enzymatic, covalently modified proteins and oxidative stress is often (although not always) involved in the mechanism of their formation. Moreover some AGEs and ALEs have the same structure, since they arise from common precursors, as in the case of carboxymethyllysine (CML) which is generated by glyoxal, which in turn is formed by both lipid and sugar oxidative degradation pathways [1]. Besides being considered as reliable biomarkers of oxidative damage, as well as predictors and prognostic factors, more recently, AGEs and ALEs have also been recognized as important pathogenetic factors of some oxidative based diseases, as supported by the following facts: 1) a strict correlation between the amount of AGEs/ALEs in tissues and fluids and disease states has been found, in both animal and human subjects; 2) a substantial amount of literature is now available reporting the molecular and cellular pathogenic mechanisms for the AGEs/ALEs involvement in the onset and progression of different oxidative-based diseases including diabetes [2], chronic renal failure [3], cardiovascular diseases [4] and neurological disorders [5]. The AGEs/ALEs damaging effect is mediated by different mechanisms, including the dysfunction of the proteins undergoing the oxidative modification, protein polymerization, signal transduction, immunoresponse and RAGE activation. Some of the biological effects are due to the loss of function of the target proteins undergoing the covalent modification, such as in the case of extracellular matrix proteins that lose their elastic and mechanical functions when modified as AGEs/ALEs and in particular, when cross-links are involved [6]. Other examples of a direct damaging effect of AGEs/ALEs can be ascribed to the covalent modification of enzymes and receptors that lose their activity due to the covalent modification involving the catalytic or binding site, or following a conformational change of the protein structure. Moreover AGEs and ALEs can be immunogenic. Hence AGEs/ALEs are now considered as promising drug targets and a substantial effort is dedicated to delve the molecular strategies aimed at preventing, reducing or removing these protein oxidation products. The different molecular approaches thus far reported can be grouped by considering at which level of the damaging AGEs/ALEs cascade they are effective and in particular if they act by inhibiting the AGEs/ALEs formation, accelerating their catabolism or blocking their biological effects. The first level of action, the inhibition of AGEs/ALEs formation, also consists of different approaches, which target the different inducers (ROS, metal ions) and intermediate products (mainly reactive carbonyl species, RCS) involved in the AGEs/ALEs formation. Antioxidants, radical scavengers, metal-ion chelators and reactive carbonyl compounds quenchers (RCS sequestering agents) represent the most promising approaches so far reported for inhibiting AGEs/ALEs formation. In some cases, as found both for natural or synthetic compounds, the inhibition of AGEs/ALEs formation does not proceed through a single specific mechanism but implicates multiple mechanisms, involving at least two of the following ones: antioxidant, radical-scavenging, metal ion chelation and RCS trapping. The second level of intervention consists of accelerating the catabolism of already formed AGEs/ALEs and this can be achieved by potentiating the endogenous proteolytic system or by using xenobiotics able to catalytically degrade AGEs/ALEs. The third level of intervention, which is also the most innovative, consists of blocking the biological response of AGEs/ALEs by inhibiting the activation of the RAGE receptors through different classes of antagonists. It should be noted that such an approach permits the blocking of the damaging effect induced not only by endogenously formed AGEs/ALEs but also by exogenously derived AGEs/ALEs. In view of a drug discovery program aim to search bioactive compounds, the first step of the research program was to set up analytical methods based on high resolution MS strategies able to test the efficacy and selectivity of compounds effective as sequestering agents of RCS and also acting as RAGE antagonists. The methods so far reported in the literature to test RCS sequestering compounds consist to measure the consumption of the aldehydes when incubated with the test compounds by a direct spectrophotometric analysis and depending on the aldehyde, the derivatization reaction should also be considered. The main limitations of such approach is that it cannot be applied to mixtures or extracts, it cannot be applied to a high throughput screening and in several cases the experimental conditions such as the acid condition of the derivatization process can dissociate the adducts between the aldehydes and the sequestering agents. Therefore, based on the limits above reported, the initial aim of this research project was focused to set-up and then apply suitable analytical methods able to screen novel RCS sequestering agents. An HPLC method was firstly set-up in order to measure the consumption of RCS (reactivity) and of endogenous carbonyls such as pyridoxal (selectivity) when incubated in presence of the tested compounds. The method was optimized in order to use a mobile phase buffered at pH 7.4 in order to avoid the acid catalyzed degradation of the formed adducts. Reactivity towards RCS was evaluated by testing the ability of the tested compound to quench 4-hydroxy-nonenal (HNE) chosen as a model of alfa,beta-unsaturated aldehydes, glyoxal (GO) and methylglyoxal (MGO) as di-carbonyl derivatives. Selectivity was tested by measuring the consumption of pyridoxal as an endogenous aldehyde. The method was firstly validated by testing the reactivity and selectivity of known RCS scavenger compounds such as edaravone (EDA), hydralazine (HY), aminoguanidine (AG), and pyridoxamine (PYR). Even though they are very effective as RCS detoxifying agents, their usage is limited due to their lack of selectivity because they react with physiological aldehydes such as pyridoxal and by a promiscuous activity: ED is a neuroprotective compound, HY an antihypertensive drug and AG a NOS inhibitor. The HPLC method was then applied to study the reactivity and selectivity of carnosine (CAR) and derivatives such as anserine, n-acetyl carnosine and of carnosine analogues designed to be stable to carnosinase, a specific metal-ion dependent homodimeric dipeptidase (carnosinase, CN1 EC.3.4.13.20),. The screening permitted to identify carnosinol, a carnosine peptidomimetic characterized by the replacement of the carboxyl group with a hydroxyl group, as a selective and reactive RCS sequestering agent, characterized by a suitable PK profile since resistant to serum carnosinase and recognized by HPEPT1 (patent application: WO2011080139). Beside evaluating the reactivity and the selectivity, the approach was then implemented by an ESI-MS approach aimed to fully characterize the covalent adducts between RCS and the sequestering agents. The investigation of the mechanisms of carbonyl quenching have so ensued a broad understanding of the biochemical mechanisms in vitro as well as in vivo that occur between cytotoxic RCS and the "sweepers" detoxifying agents. The method was then applied to test the RCS sequestering activity of proteinogenic histidine-containing dipeptides with the C-terminus capped by a methyl ester or by a primary amido group so as to study dipeptides which should be still recognized by peptide transporters and resistant to proteolysis. Moreover, the study considered diastereoisomeric pairs of dipeptides produced by alternating the absolute configuration of the histidine residue thus revealing the effect of configuration on the quenching activity (7). The second step of the work was to set-up a rapid and accurate method for testing the ability of RCS sequestering agents to inhibit protein carbonylation. To set-up the method, HNE, which is one of the most abundant and reactive lipid-derived RCS, was used as RCS, and ubiquitin [8] as a model of protein substrate. Going into details, ubiquitin was selected as a protein target since it is commercially available at high purity and at a reasonable cost and because its molecular weight is suitable for intact protein analysis using a high resolution mass spectrometer such as the Orbitrap. Despite lacking cysteine residues, ubiquitin exposes a set of reactive lysines and a highly reactive histidine, which make this protein a suitable benchmark to test protein carbonylation and its inhibition. The method was firstly validated by using known RCS sequestering agents and the results well correlate with those obtained by HPLC analysis. The method was found suitable to test mixtures or extracts and to be used for HTS applications. AGEs and most probably ALEs are ligands and activators of the receptor RAGE whose activation is involved in a NF-kB pathway, leading to NADPH oxidase activation, oxidative stress and a condition of inflammatory and pro-fibrotic response. Since the damaging AGEs-RAGE axis has been associated to the onset and/or progression of many chronic and degenerative oxidative based disease and in many metabolic disorders, it has been recently hypothesized that the AGE-RAGE axis represents a promising drug target. One approach to counteract the AGEs-RAGE axis is represented by the design of RAGE antagonists. It should be considered that a drug design approach aimed to screen RAGE antagonists firstly requires a reliable analytical method able to test the binding affinity of small molecules and the set-up of such a method represents the aim of the present research program that is based on the following steps: \uf0d8 RAGE expression as recombinant protein in E. coli strain ORIGAMI B (DE3) with recombinant plasmid pET-15b VC1; \uf0d8 Application of the automated loop injection High Resolution Mass Spectrometry (MS) method, based on the High Resolution Mass Spectrometry method developed to study the Ubiquitin-RCS interaction, to inquire in vitro non-covalent binding relationship between low molecular weight AGEs and recombinant RAGE. The receptor for advanced glycation end products (RAGE) is a type I transmembrane glycoprotein of the mmunoglobulin superfamily of cell surface receptors. RAGE extracellular portion is involved in ligand binding and contains one \u201cV\u201d \u2013type followed by two \u201cC\u201d-type immunoglobulin-like domains (V-C1-C2 structure). V-C1 domains form an integrated unit whereas C2 domain is attached to V-C1 by a flexible linker but is a fully independent unit. As a general strategy, we decided to express the V-C1 portion of the extracellular domain of human RAGE (sRAGE) as protein target since it is water soluble and involved in the molecular recognition and engagement of the AGEs ligands. Therefore, V, V-C1 and s-RAGE expression was focused on the recombinant protein expression carried out by the protein studies obtained through the E. coli expression system. Despite a greater facility to handling the microorganism, E. coli in contrast to eukaryotic expression system, is not capable to perform post-translational modifications, including glycosylation, which are instead present in the human RAGE. Regardless for the lack of this peculiarity, numerous studies reported in literature, indicate that, post-translational modifications have not significantly effect on the RAGE properties regarding for instance the bind activity toward certain ligands and so that, the bacterial E. coli expression system represent one of the most used host for the heterologous proteins expression. A great advantage by using E.coli is its peculiarity of secreting the protein of interest in the medium that it can be easily purified in one-step. This advantage have avoided the use of tags, such as the Poly-His, that could affect the property of sRAGE during high throughput screening of libraries of compounds and making unnecessary the removal step by specific protease treatments. The high resolution mass spectrometric (ESI-MS) approach in top-down and bottom-up approach was then used in order to verify the identity of the protein. The multi-charged ESI-MS spectrum of the recombinant protein corresponding deconvoluted MS spectrum showing the MW of 24580 Da which is consistent with the theoretical one. GSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPV-PLEEVQLVVE. The primary sequence was then identified by a bottom-up approach consisting to enzymatically digest the protein, separate the peptides by reversed phase capillary column. Eluted peptides were then sequenced by MS/MS analyses. The primary structure of the protein corresponding to the predicted sequence on the bases of the designed nucleotide sequence of sRAGE. After having characterized the protein, a MS approach to study the non covalent binding of RAGE with ligands was set-up. The ligand-binding properties of RAGE was studied by a native MS method that is suitable to study the non covalent interactions between ligands and recombinant V-C1. The advantages of native MS over other methods is that it does not require labeled target or ligands, it is characterized by high sensitivity, low sample consumption, fully automatation and that ligands can be screened as mixture. The experiments consist in maintaining the concentration of VC1 constant and increasing the ligand concentration. The ligands used are well known low molecular weight sRAGE ligands such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) derived peptide [9]. Unmodified peptides were also used as controls. Validation of the method was obtained by comparing the Kd values obtained by native MS analysis in respect to the Kd values determined from fluorescence titration experiments. The native MS method was found accurate and suitable to test libraries in order to understand the structure requirements for RAGE recognition as well as for searching antagonists. References [1] Baynes JW . Chemical modification of proteins by lipids in diabetes. Clin Chem Lab Med 2003 ; 41 : 1159 \u2013 1165. [2] Yamagishi S , Maeda S , Matsui T , Ueda S , Fukami K , Okuda S . Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012 ; 1820 : 663 \u2013 671. [3] Iacobini C , Menini S , Ricci C , Scipioni A , Sansoni V , Mazzitelli G , et al . Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptormediated mechanisms . J Pathol 2009 ; 218 : 360 \u2013 369. [4] Del Turco S , Basta G . An update on advanced glycation endproducts and atherosclerosis. Biofactors 2012 ; 38 : 266 \u2013 274. [5] Li JL , Liu DN , Sun L , Lu Y , Zhang Z . Advanced glycation end products and neurodegenerative diseases: Mechanisms and perspective. J Neurol Sci 2012 ; 317 : 1 \u2013 5. [6] Ciulla MM , Paliotti R , Carini M , Aldini G . Fibrosis, enzymatic and non-enzymatic cross-links in hypertensive heart disease. Cardiovasc Hematol Disord Drug Targets 2011. [7] Vistoli G, De Maddis D, Straniero V, Pedretti A, Pallavicini M, Valoti E, Carini M, Testa B, Aldini G. Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents. Eur J Med Chem. 2013 Aug;66:153-60 [8] Liang X, Chen Y, Zhuang J, Zhang M, Xiong W, Guo H, Jiang F, Hu P, Guo D, and Shi W, Advanced oxidation protein products as prognostic biomarkers for recovery from acute kidney injury after coronary artery bypass grafting. Biomarkers 17 (2012) 507-12. [9] Xue J, Rai V, Singer D, Chabierski S, Xie J, Reverdatto S, Burz DS, Schmidt AM, Hoffmann R, Shekhtman A. Advanced glycation end product recognition by the receptor for AGEs. Cell Press. 2011 May 11;19(5):722-32

Molecular mechanism of poly(ADP-ribosyl)ation catalyzed by human poly(ADP-ribose) polymerase-1

Human poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme which catalyzes protein poly(ADP-ribosyl)ation upon binding to DNA. NAD+ is used as a co-substrate in the reaction via iterative transfer of its ADP-ribose moiety to acceptor proteins including PARP-1 itself, yielding elongated and branched poly(ADP-ribose) (PAR) polymers. This type of protein posttranslational modification has been demonstrated in the regulation of diverse biological processes including DNA repair, gene expression, cell cycle, etc. Therefore, elucidating the catalytic mechanism of PARP-1 would not only advance our understanding of how its enzymatic activity is regulated under physiological and pathophysiological conditions, but also greatly benefit the development of novel therapeutics involving pharmacological manipulation of PARP-1. In this dissertation, the molecular mechanism of DNA-dependent poly(ADP-ribosyl)ation by human PARP-1 was addressed from an enzymological perspective in terms of the allosteric ligand DNA, the substrate NAD+, and the PARP-1 protein–DNA complex as a whole. By site-specific labeling of the DNA-binding domain AB of PARP-1 and DNA ligands with fluorophores, quantitative binding kinetics of AB with DNA was investigated by single-molecule fluorescence spectroscopy. Two binding modes, one involving a strongly-associated protein–DNA complex and the other being transient, were suggested by the experimental data. To probe the catalytic mechanism of the initiation, elongation, and branching step of poly(ADP-ribosyl)ation with regard to NAD+ substrate scope, analogues of NAD+ with fluoro-substituted ribose ring were synthesized chemoenzymatically and employed as substrates. The results are consistent with the proposed mechanism that the ADP-ribosyl transfer reaction proceeds through an oxocarbenium-like transition state. Mass spectrometry and biochemical approaches were utilized to decipher the poly(ADP-ribosyl)ation sites on PARP-1 and the chemical nature of PAR–protein linkages. The data confirm the existence of automodification sites beyond domain D, and lysine could be the targeted residue for poly(ADP-ribosyl)ation, either enzymatically or nonenzymatically. The macromolecular mechanism of DNA-dependent PARP-1 automodification was established by an in vitro radioactivity-based poly(ADP-ribosyl)ation assay using structurally distinguishable PARP-1 mutants. The data support the model of an intermolecular process. Top-down MS analysis and crosslinking assay bolster the monomeric structure of domain C in solution and its participation in interdomain contacts during PARP-1 catalysis. Taken together, these mechanistic studies provide further insight into the catalytic strategies exploited by human PARP-1 complementary to recent reports of structural characterization, and may help discover better therapeutic agents modulating poly(ADP-ribosyl)ation.Pharmaceutical Science

PREDICTING THE PHYSICOCHEMICAL PROPERTIES OF PORK BELLY AND THE EFFECT OF COOKING AND STORAGE CONDITIONS ON BACON SENSORY AND CHEMICAL CHARACTERISTICS

The first objective of this research was to use a widely varying pig population to create prediction algorithms for dual energy X-ray absorptiometry (DXA) pork carcass compositional estimate and pork belly softness measurement. Further, bellies with compositional extremes were used in bacon production and cooked in two ways to determine the impact of composition, storage days and cooking method on lipid and protein oxidation as well as heterocyclic aromatic amines. A total of 648 pigs, either barrows or gilts, from three sire breeds (Lacombe, Duroc or Iberian boar × Large White * Landrace F1 dams), were provided one of three diets (conventional, canola-based or flaxseed-based feed) ad libitum until they reached either ~120 or 140 kg slaughter weight. These variations were intentionally introduced so that the animal population could adequately represent the variation applicable to commercial production. Following slaughter, carcass sides and primal cuts were scanned under DXA equipment. For the second experiment, 198 left side bellies were assigned to belly-flop angle and subjective score measurements to evaluate pork belly softness. The third experiment employed 44 right side bellies which were randomly selected from the treatment extremes (barrows or gilts, Iberian or Lacombe, and control or flaxseed based diet). These 44 bellies were processed into bacon slices which were cooked with either microwave heating or pan frying after 2 or 28 days of refrigerated storage. Regardless of variation in animal population, DXA accurately predicted dissected/chemical fat and lean content of carcass sides and primal cuts (R2 > 0.94, P 0.05). The cooking treatments and storage days also had minimal effects on bacon sensory attributes. Overall, the present study established mathematical models to improve DXA estimate of pork carcasses and enhance pork belly softness assessments. The results could also inform public health recommendations regarding choice of cooking method for bacon

PHYSICOCHEMICAL AND FUNCTIONAL PROPERTY MODIFICATION OF MYOFIBRILLAR PROTEIN BY PHENOLIC COMPOUNDS UNDER OXIDATIVE STRESS

Polyphenol-rich spices and extracts of phenolic compounds are widely utilized in meat processing to modify product flavors. Chemically, polyphenols are reactive with myofibrillar protein (MP), the most functional fraction of all muscle proteins responsible for texture development in comminuted meat products. Such protein–polyphenol interaction is prevalent under oxidative conditions that are common in meat processing. As a large group of phytochemicals with diverse structures, phenolic compounds are known to interact with MP with varying efficacies. Yet, the structure-function relationship of polyphenols in eliciting modification of MP is poorly understood. The overall objective of this dissertation research was to elucidate the effect of structurally related phytophenols on the physicochemical properties of MP and resultant changes in protein functionalities, i.e., gelation and emulsification. To establish appropriate testing conditions, a mild oxidative environment was introduced using glucose oxidase (GOx), and the simplest phenolic compound, gallic acid (GA), was used to investigate the effect on the physicochemical and gelling behavior of MP. Compared with non-oxidized (control) MP, GOx-mediated oxidation facilitated both covalent and noncovalent interactions between GA (6, 30, and 60 μmol/g protein) and protein through promoting protein structural unfolding. Such modifications significantly enhanced the gelling capacity of MP, which was evidenced by up to 86% and 53% increases (P \u3c 0.05) in gel elasticity (G′) and breaking strength, respectively. Based on the above observations, six structurally related monophenolic acids varying in hydroxyl substitution and sidechain groups, i.e., GA, syringic acid (SA), coumaric acid (CMA), caffeic acid (CFA), ferulic acid (FA), and chlorogenic acid (CA), were examined for their effects on MP conformation and gelation under GOx oxidative stress. The elasticity and breaking strength of MP gels were markedly enhanced by all phenolic acids, of which GA and CA induced the highest final G′ values of 291 and 281 Pa (P \u3c 0.05), respectively, as compared with 214 Pa of the control MP sample without phenolic addition. Different reaction modes were evident for these two most effective phenolic acids in improving protein gelation. With the least structural hinderance, the smallest GA facilitated protein cross-linking through covalent adduction to amino acid sidechains. On the other hand, having a bulky sidechain group, CA was the most effective in promoting protein unfolding due to the multiple functional groups, including 5 hydroxyl groups and 1 extra hydrocarbon ring (quinic acid). The findings of structure-dependency of phenolic activity prompted the following experiment where phenolic compounds with more than one phenol structures were included to investigate their influence on MP functionalities. Here, in addition to three monophenols, i.e., GA, CA, and propyl gallate (PG), two diphenols, i.e., quercetin (QT) and catechin (CC), and one triphenol, i.e., (–)-epigallocatechin-3-gallate (EGCG), were selected to further explore the structure-activity relationship of phenolic compounds on MP functionalities under GOx oxidation. MP-stabilized oil-in-water emulsions were prepared to assess protein emulsifying properties, and an emulsion-filled composite gel system was adopted as a model to mimic comminuted meat products in which MP acted as both an emulsifier and a building block for the protein matrix within the gel. In the emulsion system, phenolic compounds with less polarity, i.e., PG, QT, and CC, significantly improved the emulsifying capacity of MP by increasing protein partition at the oil-water interface by 15, 17, and 23%, respectively (P \u3c 0.05). In the MP–emulsion composite gel system, all three monophenols (GA, CA, and PG) and the diphenol QT increased the MP gel strength to a greater extent than CC (diphenol) and EGCG (triphenol). The flavanol structure in CC appeared to interfere with gel structure development. The multiple phenol structures in EGCG caused protein aggregation so severe that both emulsifying and gelling properties of MP were weakened. Lipid oxidation was retarded by all phenols in MP–emulsion composite gels during storage at 4 °C for 7 days with PG and QT being the most effective. The above findings established that the type and size of the sidechain groups, the number of hydroxyl attached to the benzene ring, as well as the number of the phenol moiety have an important role in affecting phytophenol–MP interaction and the protein functionality under oxidative condition. Small-sized phenolic compounds tend to promote MP gelation and emulsification, and larger sized (such as EGCG) exhibited negative effects due to the propensity to facilitate extensive protein aggregation